DESCRIPTION: This proposal will investigate molecular and cellular mechanisms involved in linking membrane depolarization to calcium release in skeletal muscle. Voltage clamped fibers from frog and rat will be used. Electrical and optical recordings will allow both measurement of voltage sensor charge movement and macroscopic calcium release. Confocal imaging of localized (unitary) calcium release and laser photorelease of caged Ca will also be employed. The first aim seeks to determine whether Ca release is activatable by Ca in skeletal muscle. The approach will be to introduce Ca transients that mimic those occurring normally to see if Ca release is activated. Conversely, the normal increase in local Ca can be perturbed by buffers to see if physiological release is altered. This will be done at both the microscopic and macroscopic levels. In the second aim, the role of calcium dependent inactivation will be tested. The kinetics of elementary events of Ca release will be analyzed to see if evidence for an inactivated state can be found. Buffers and caged calcium release will also be used to investigate this possibility. In the third aim, possible heterogeneous behavior of Ryas will be investigated. It will be determined whether the observed behavior of "eager triads" that seem especially susceptible to activation result from some idiosyncrasy of the experimental procedures or whether they contain more responsive channels. In Aim 4, the role of a hypothetical Ca binding site near the voltage sensor that affects charge movement will be investigated. Photorelease of Ca will be used to mimic the normal Ca release flux and its effect on charge movement determined. In the final aim, optical measurements will be used to localize DHPR responsible for Ica and voltage sensing to determine if separate molecules subserve these two functions.